Application of METS1 gene in regulating grain type and 1000-grain weight of rice

Abstract

The application discloses METS1 The application discloses application of a gene in regulating rice grain type and 1000-grain weight, and belongs to the technical field of genetic engineering. METS1 The application finds that overexpression of the gene can significantly reduce rice grain length, grain width and 1000-grain weight, and the reduction effect is negatively correlated with the expression amount of the gene. METS1 Knocking out the gene can significantly increase the rice grain length and 1000-grain weight. METS1 The application provides a new regulation gene for early revealing the regulation mechanism of the rice grain type and 1000-grain weight. METS1 The gene can be used in genetic engineering breeding of different grain type rice, and has important significance for accelerating the breeding process and reducing the breeding workload.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to... METS1 Application of genes in regulating rice grain shape and thousand-grain weight. Background Technology

[0002] Grain output is an important indicator for measuring a country's agricultural development level and its ability to ensure a stable and secure food supply. Increasing yield per unit area means obtaining greater returns with the same land and cost inputs. It is an important measure to increase total grain output and ensure food supply without increasing the area under grain cultivation.

[0003] The length, width, and thousand-grain weight of rice seeds directly affect the yield, quality, economic benefits, and industrial competitiveness of rice. Thousand-grain weight, the weight of 1000 mature rice grains, is one of the most critical indicators for measuring rice yield. The formula for calculating yield per unit area is: Yield per unit area = Number of effective panicles × Number of filled grains per panicle × Thousand-grain weight. When the number of effective panicles and the number of filled grains per panicle remain constant or are difficult to increase significantly, increasing the thousand-grain weight is one of the most direct and effective ways to increase rice yield per unit area. Increased grain length and width directly lead to an increase in the weight of a single rice grain, which ultimately manifests as an increase in thousand-grain weight.

[0004] In addition, the length and width of rice grains directly determine the marketability of rice. Long-grain rice (such as indica rice and fragrant rice) is generally more popular in the international market, and is considered to have a better taste and a higher price. Grain width is also related to chalkiness (the opaque white part in the center of the rice grain). Increasing the grain width helps to reduce chalkiness, making the rice grains look more crystal clear, with a better commercial appearance and a higher price. Summary of the Invention

[0005] The purpose of this invention is to provide METS1 The application of this gene in regulating rice grain shape and thousand-grain weight aims to provide a new candidate gene that has a significant regulatory effect on rice grain shape and thousand-grain weight, contributing to the early elucidation of the regulatory mechanism of rice grain shape and thousand-grain weight and improving rice yield and quality.

[0006] To achieve the above objectives, the present invention provides METS1 Application of genes in regulating rice grain shape METS1 The CDS sequence of the gene is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

[0007] Preferably, the rice grain shape refers to the length and width of the rice grain.

[0008] Preferred, overexpression METS1 The gene significantly reduced rice grain length and width, and the reduction effect was similar to... METS1The expression level of the gene was negatively correlated; knocking out the gene significantly increased the grain length of rice.

[0009] Including the above METS1 Application of vectors containing CDS sequences of genes in regulating rice grain shape.

[0010] Including the above METS1 Application of strains containing CDS sequences of genes in regulating rice grain shape, wherein the strains are Escherichia coli and Agrobacterium.

[0011] METS1 Application of genes in regulating the thousand-grain weight of rice METS1 The CDS sequence of the gene is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4; METS1 Gene expression levels are negatively correlated with the thousand-grain weight of rice.

[0012] Including the above METS1 The application of strains containing the CDS sequence of genes in regulating the thousand-grain weight of rice, wherein the strains are Escherichia coli and Agrobacterium.

[0013] Therefore, the present invention provides METS1 The specific technical effects of gene application in regulating rice grain shape and thousand-grain weight are as follows:

[0014] (1) This invention is the first to discover overexpression METS1 The gene significantly reduces rice grain length, grain width, and thousand-grain weight, and the reduction effect is comparable to... METS1 The expression levels of the gene were negatively correlated; knocking out this gene significantly increased rice grain length and thousand-grain weight; this provides a new regulatory gene for revealing the regulatory mechanisms of rice grain shape and thousand-grain weight as soon as possible.

[0015] (2) Those skilled in the art can examine METS1 Gene expression levels are used to assess rice grain shape and thousand-grain weight. METS1 Genes can be used in genetic engineering breeding of rice with different grain types, which is of great significance for accelerating the breeding process and reducing the workload of breeding. The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1This is the electrophoretic detection result of a single transgenic plant with a hyg-positive band in Example 3 of this invention.

[0018] Figure 2 These are the statistical results of the relative expression levels of the recipient parent rice GH998 and the transgenic plants in Example 4 of this invention; a, b, and c represent the expression levels in... P The difference was significant at the <0.05 level.

[0019] Figure 3 These are photographs of the recipient parent rice GH998 and the transgenic plant in Example 5 of this invention; where A is the length of 10 rice grains; B is the width of 10 rice grains; the scale bar in the figure is 1 cm.

[0020] Figure 4 This is a statistical result of the grain shape and thousand-grain weight of rice from the recipient parent rice GH998 and the transgenic plants in Example 5 of this invention; where A represents the grain length statistical result; B represents the grain width statistical result; C represents the thousand-grain weight statistical result; and a and b in the figure represent the... P The difference was significant at the <0.05 level.

[0021] Figure 5 These are photographs of the recipient parent rice Nipponbare and its knockout mutant in Example 6 of this invention; where A is the length of 10 rice grains; B is the width of 10 rice grains; the scale bar in the figure is 1 cm.

[0022] Figure 6 This is a statistical result of the grain shape and thousand-grain weight of rice from the recipient parent rice Nipponbare and its knockout mutant in Example 6 of this invention; where A is the statistical result of grain length; B is the statistical result of grain width; C is the statistical result of thousand-grain weight; and a and b in the figure represent the results of... P The difference was significant at the <0.05 level.

[0023] Figure 7 This is the sequencing alignment result of the mutant target in Example 6 of the present invention. Detailed Implementation

[0024] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0025] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0026] All instruments, equipment, and reagents used in the examples were obtained commercially; methods not described in detail are conventional techniques in the art; information on the culture media used in the examples is as follows:

[0027] N6 solid screening medium with 150 mg / L hygromycin: Hygromycin was added to N6 solid medium to make the mass concentration of hygromycin 150 mg / L;

[0028] N6 solid screening medium with 200 mg / L hygromycin: Hygromycin was added to N6 solid medium to make the mass concentration of hygromycin 200 mg / L;

[0029] Differentiation medium: 6-BA 2mg, NAA 0.2mg, N6 4g, hydrolyzed casein 1g, inositol 0.1g, sucrose 25g, sorbitol 2.4g, agar powder 7g, deionized water 1L.

[0030] Example 1

[0031] get METS1 The CDS sequence of the gene is as follows:

[0032] S11 was obtained by PCR amplification using DNA from common wild rice Y11 (Oryzarufipogon Griff.) as a template, with primers primer1 (sequence shown in SEQ ID NO.1) and primer2 (sequence shown in SEQ ID NO.2). METS1 The CDS sequence of the gene, the PCR system, and the amplification program were configured / set according to the instructions accompanying the high-fidelity enzyme.

[0033] SEQ ID NO.1: ATGGCGTCTCACATTG

[0034] SEQ ID NO.2: TCACTTCGCG CTGGCG

[0035] S12. Perform agarose gel electrophoresis on the PCR products obtained in S11, and then use a kit to recover the gel. Ligate the recovered products into the Zero sequencing vector (the ligation method is in accordance with the instructions accompanying the Zero sequencing vector) to obtain the recombinant vector. Then, transform the obtained recombinant vector into DH5α competent cells, select positive clones, and perform sequencing.

[0036] Sequencing results showed that the sequence of the PCR product obtained from S11 was as shown in SEQ ID NO.3, with a length of 2301 bp, and it was named... METS1 (Methionine synthase 1, LOC_Os12g42876) gene.

[0037] SEQ ID NO.3:

[0038]

[0039] SEQ ID NO.4:

[0040] MASHIVGYPRMGPKRELKFALESFWDGKSSAEDLEKVATDLRASIWKQMADAGIKYIPSNTFSYYDQVLDTAAMLGAVPERYSWTGGEIGFSTYFSMARGNATVPAMEMTKWFDTNYHFIVPELGPNTKFSYS SHKAVNEYKEAKALGVDTVPVLVGPVSYLLLSKPAKGVEKSFALLSLLSSILPVYKEVIAELKAAGATWIQFDEPTLVLDLDSHQLAAFSAAYTELESALSGLNVLIETYFADIPAESYKTLTSLNSVTAYGF DLIRGAKTLDLIKSAGFPSGKYLFAGVVDGRNIWADDLAASLTTLESLEAIVGKDKLVVSTSCSLMHTAVDLVNETKLDSEIKSWLAFAAQKVVEVNALAKALAGQKDEAYFAANAAAQASRRSSPRVTNEEV QKAAAALKGSDHRRATNVSARLDAQQKKLNLPVLPTTTIGSFPQTVELRRVRREYKAKKISEEEYISAIKEEISKVVKIQEELDIDVLVHGEPEETIWLSTSVSSFLVLHSLPTDGCNLMDHGVSSHQLSMVM

[0041] Example 2

[0042] Build METS1 Gene overexpression vectors are as follows:

[0043] S21. Using cDNA from wild rice Y11 (Oryzarufipogon Griff.) as a template, PCR amplification was performed using primers primer1 and primer2 from Example 1. The PCR system and amplification program were configured / set according to the instructions accompanying the high-fidelity enzyme.

[0044] The amplification products were subjected to agarose gel electrophoresis, and then the gel was recovered using a kit. The recovered products were ligated into the vector Zero (according to the instructions accompanying the vector) to obtain a positive clone of recombinant Zero-METS1. The recombinant vector Zero-METS1 was digested with restriction endonucleases Kpn I and Pac I to obtain the OE-METS1 fragment.

[0045] S22. Digest the expression vector PMDC32 with restriction endonucleases Kpn I and Pac I to obtain the linear expression vector PMDC32. Recover the linear fragment using a kit. Integrate the OE-METS1 fragment obtained in step S21 into the linear expression vector PMDC32 using homologous recombination directional cloning (refer to the PMDC32 instruction manual for specific methods) to obtain the recombinant expression vector PMDC32-OE-METS1.

[0046] The obtained recombinant expression vector PMDC32-OE-METS1 was sent to the company for sequencing. The results showed that the recombinant expression vector inserted the nucleotide sequence shown in SEQ ID NO.1 in the forward direction at the Kpn I restriction site of the expression vector PMDC32, that is, the DNA sequence between the Kpn I and PacI recognition sites (recognition sequence) of PMDC32 was successfully replaced with the DNA sequence shown in SEQ ID NO.1.

[0047] Example 3

[0048] The genetic transformation of the recombinant expression vector PMDC32-OE-METS1 is detailed below:

[0049] S31. Transformation: The recombinant expression vector PMDC32-OE-METS1 obtained in Example 2 was transformed into DH5α competent cells using the heat shock method. The specific steps were performed according to the instructions accompanying the DH5α competent cells.

[0050] S32. For bacterial culture PCR verification, the product was spread on LB medium containing 50 ng / μL kana and incubated overnight at 37°C. Then, a single colony was picked in a clean bench as a template and PCR amplification was performed using the m13 universal primer. The amplification system and amplification program were configured / set according to the instructions attached to the Taq DNA polymerase.

[0051] S33. Plasmid extraction: Take positive clones and shake them to form bacteria. Then, use a kit to extract the plasmid and detect the concentration to obtain the recombinant expression vector PMDC32-OE-METS1.

[0052] S34. Plasmid transformation: The recombinant expression vector PMDC32-OE-METS1 obtained in step S33 is introduced into Agrobacterium tumefaciens EHA105 by heat shock to obtain recombinant Agrobacterium tumefaciens EHA105 containing the recombinant expression vector PMDC32-OE-METS1.

[0053] Recombinant Agrobacterium tumefaciens EHA105 containing the recombinant expression vector PMDC32-OE-METS1 was cultured at 28℃ for 16 h. The bacterial cells were collected and diluted using N6 liquid medium (Sigma, catalog number C1416) containing 100 μM acetylsyleugenol to obtain a diluted bacterial solution. The solution was then diluted to the OD value. 600 It is approximately 0.5.

[0054] S35. Infection: The embryogenic callus tissue of mature embryonic GH998 indica rice cultured for one month was mixed with the diluted bacterial solution obtained in step S34 and infected for 30 minutes. After the bacterial solution was dried with filter paper, it was transferred to N6 solid co-culture medium and co-cultured at 24℃ for 3 days to obtain the co-cultured callus tissue.

[0055] S36. Screening: The callus tissue after co-culture treatment in step S35 is inoculated onto N6 solid screening medium containing 150 mg / L hygromycin for the first screening.

[0056] On day 16 of the first screening, healthy callus tissue was picked and transferred to N6 solid selection medium containing 200 mg / L hygromycin for a second screening. Subculture was performed every 15 days for a total of 1 subculture to obtain anti-callus tissue.

[0057] S37. Differentiation culture yields overexpression-positive plants. The resistant callus obtained in step S36 is selected and transferred to a differentiation medium containing 150 mg / L hygromycin for differentiation. The plants are cultured at 24℃ for 45 days (at which point the aboveground part of the plant is about 15 cm tall). The bottle opening is opened for hardening off for 3 days, and then the plants are transplanted to a greenhouse for cultivation. These are the PMDC32-OE-METS1 plants (referred to as T0 generation).

[0058] Different transformation events (transformed into different callus tissues) were named N-OE1, N-OE2, etc., respectively, representing the overexpression positive plants transformed into the recombinant vector PMDC32-OE-METS1. The corresponding plants were named METS1-OE1, METS1-OE2, etc.

[0059] S38, Turn METS1 For PCR identification of the gene-producing plants, genomic DNA was extracted from the leaves of T0 generation seedlings of METS1-OE1 and METS1-OE2 obtained in step S37 and seedlings of the recipient parent rice GH998. PCR was performed using primers hyg-F (sequence shown in SEQ ID NO. 5) and hyg-R (sequence shown in SEQ ID NO. 6). The PCR system and amplification program were configured / set according to the instructions accompanying the Taq enzyme. The PCR products were subjected to agarose gel electrophoresis. Single plants showing a positive hyg band (900 bp) were identified. Figure 1 Two transgenic plants were obtained, identified as transgenic single plants.

[0060] SEQ ID NO.5:AAAAGTTCGACAGCGTCTCCGACC

[0061] SEQ ID NO.6: TCTACACAGCCATCGGTCCAGACG

[0062] Example 4

[0063] Identification of the transfer obtained in Example 3 METS1 In genetically modified plants METS1 The gene expression levels are as follows:

[0064] RNA was extracted from the leaves of the transgenic plants obtained in Example 3 and the recipient parent rice GH998. Actin was set as the internal control. Using the internal control primers Actin-F (sequence shown in SEQ ID NO. 7) and Actin-R (sequence shown in SEQ ID NO. 8), and... METS1 The gene-specific quantitative primers METS1-qRT-F (sequence shown in SEQ ID NO. 9) and METS1-qRT-R (sequence shown in SEQ ID NO. 10) were used to perform real-time PCR to detect the gene-specific PCR results in different transgenic plants. METS1 Changes in gene expression levels.

[0065] Among them, the relative expression levels of the two transgenic plants with the greatest difference from the recipient parent rice were as follows: Figure 2 As shown, the expression level of the METS1 gene in positive plants transformed with the recombinant vector PMDC32-OE-METS1 was significantly higher than that in the control plants (recipient parent rice GH998). METS1 A significant increase in gene expression levels.

[0066] SEQ ID NO.7: ATTTGGCACCACACATTCTAC

[0067] SEQ ID NO.8: ATAACCTTCGTAGATTGGGACT

[0068] SEQ ID NO.9: ACAAGACCCTCACATCCCTG

[0069] SEQ ID NO. 10: AGTGAGCAGGAAGTCGAGAC.

[0070] Example 5

[0071] Identification and transfer METS1 The phenotypes of the genetically modified plants are as follows:

[0072] METS1-OE1, METS1-OE2 plants and the recipient parent rice GH998 were planted at the Hainan experimental base to observe the differences in grain type phenotype between METS1-OE1, METS1-OE2 plants and GH998 throughout the entire growth period.

[0073] The measurement and observation results are shown in Table 1. Figure 3 and Figure 4 As shown, compared with GH998 plants, METS1-OE1 and METS1-OE2 plants both exhibited significantly lower grain length, grain width, and thousand-grain weight compared with the control group GH998. P <0.05), proof METS1 Genes are involved in controlling the grain shape and yield of rice.

[0074] Table 1. Statistical results of rice grain length, grain width, and thousand-grain weight

[0075]

[0076] Example 6

[0077] The knockout vector was constructed and genetically transformed into Nipponbare, as detailed below:

[0078] Using CRISPR / Cas9 technology, a target (sequence shown in SEQ ID NO.11) was designed for knockout in Nipponbare. METS1 The gene and mutant seeds showed a significant increase in seed length and thousand-seed weight. Figure 5 , Figure 6 Using primers based on the sequences shown in SEQ ID NO.12 and SEQ ID NO.13, the mutation status of the target site was detected, and an insertion of base A was detected at the target site. Figure 7 ),illustrate METS1 Genes negatively regulate rice grain shape and yield.

[0079] SEQ ID NO.11: GAATCAGATCGTCCTGGTGCTGG

[0080] SEQ ID NO.12: TGGATCTGAGTAATCCTGTTAGCT

[0081] SEQ ID NO.13: AAATCCTCAGCGCTGCTCTT

[0082] Therefore, this invention is the first to discover overexpression METS1 The gene significantly reduces rice grain length, grain width, and thousand-grain weight, and the reduction effect is comparable to... METS1The expression levels of these genes are negatively correlated; this provides new regulatory genes for the early elucidation of the regulatory mechanisms of rice grain shape and thousand-grain weight; those skilled in the art can further investigate... METS1 Gene expression levels are used to assess rice grain shape and thousand-grain weight. METS1 Genes can be used in genetic engineering breeding of rice with different grain types, which is of great significance for accelerating the breeding process and reducing the workload of breeding.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. METS1 The application of genes in regulating rice grain shape is characterized by: METS1 The CDS sequence of the gene is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4; The rice grain shape refers to the length and width of the rice grain; overexpression METS1 The gene significantly reduced rice grain length and width, and the reduction effect was similar to... METS1 Gene expression levels are negatively correlated.

2. The application according to claim 1, characterized in that: The overexpression METS1 The genetic approach is to use methods that include METS1 Rice genetic transformation was performed using an overexpression vector of the gene CDS sequence.

3. The application according to claim 2, characterized in that: The overexpression METS1 The gene was expressed by infecting rice with a strain containing the overexpression vector; the strain was Agrobacterium.

4. METS1 The application of genes in regulating the thousand-grain weight or grain length of rice is characterized by: METS1 The CDS sequence of the gene is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4; knockout METS1 The gene significantly increases the thousand-grain weight or grain length of rice.

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